Federal Register / Vol. 45, No. 20 / Tuesday, January 29, 1980 / Notices 
6735 
III— A— 2— a— (2) — (c)— {^)— (o). Pi physical 
containment + an.HVl host-vector shall 
be used for DNA recombinants 
produced with purified subgenomic 
cDNA segments. ( 38 ) 
lII-A-2-a-(2)-(c)-(2)-(6). P2 physical 
containment-t-an HVl host and a vector 
certified for use in an HV2 system, or 
P3 + HV1, shall be used for DNA 
recombinants produced with (i) cDNA 
copies of the whole genome, or (ii) 
purified cDNA copies of viral mRNA. 
[37) 
III— A— a— A— (2)— ( d ) . Double-Stranded 
Segmented RNA Viruses. Pi physical 
containment + an HVl host-vector shall 
be used for DNA recombinants 
produced with (i) mixtures of 
subgenomic cDNA segments, (ii) a 
specific subgenomic cDNA segment, or 
(iii) purified cDNA copies of viral 
mRNA. ( 37) 
III— A— 2— a— (2)— (e). RNA Plant Viruses 
and Plant Viroids. Pi physical 
containment + an HVl host-vector shall 
be used for DNA recombinants 
produced with (i) cDNA copies of the 
whole viral genone, (ii) subgenomic 
cDNA segments, or (iii) purified cDNA 
copies of viral mRNA. [37) 
III— A— 2— a— (3). Intracellular Viral 
DNA. Physical and biological 
containment specified for shotgun 
experiments with eukaryotic cellular 
DNA [see Section III— A— (1)— (a)] shall be 
used for DNA recombinants produced 
with integrated viral DNA or viral 
genomes present in infected cells. 
III-A-2-b. Eukaryotic Organelle 
DNAs. P2 physical containment + an 
HVl host-vector, or P1 + HV2, for 
mitochondrial or chloroplast DNA from 
eukaryotes when the organelle DNA has 
been obtained from isolated organelles. 
Otherwise, the conditions given for 
shotgun experiments apply. 
lII-A-2-c. Prokaryotic Plasmid and 
Phage DNAs. The containment levels 
required for shotgun experiments with 
DNA from prokaryotes apply to their 
plasmids or phages (See Section III— A— 
1— b). 
Ill— A— 3. Lowering of Containment 
Levels for Characterized or Purified 
DNA Preparations and Clones. Many of 
the risks which might conceivably arise 
from some types of recombinant DNA 
experiments, particularly shotgun 
experiments, would result from the 
inadvertant cloning of a harmful 
sequence. Therefore, in cases where the 
risk of inadvertently cloning the 
“wrong" DNA is reduced by prior 
enrichment for the desired piece, or in 
which a clone made from a random 
assortment of DNAs has been purified 
and the absence of harmful sequences 
established, the containment conditions 
for further work may be reduced. The 
following section outlines the 
mechanisms for-such reductions. 
III-A-3-a. Purified DNA Other than 
Plasmids, Bacteriophages, and Other 
Viruses. The formation of DNA 
recombinants from cellular DNAs that 
have been purified (47) and in which the 
absence of harmful sequences has been 
established (J) can be carried out under 
lower containment conditions than used 
for the corresponding shotgun 
experiment. [42) The containment may 
be decreased one step in physical 
containment (P4— »P3; P3— »P2; P2— >Pl) 
while maintaining the biological 
containment specified for the shotgun 
experiment, or one step in biological 
containment (HV3— HV2; HV2— HVl) 
while maintaining the specified physical 
containment. The institutional biosafety 
committee (1BC) must review such a 
reduction and the approval of the IBC 
and of the NIH must be secured before 
such a reduction may be put into effect. 
IlI-A-3-b. Characterized Clones of 
DNA Recombinants. When a cloned 
DNA recombinant has been rigorously 
characterized and the absence of 
harmful sequences has been established 
[3), experiments involving this 
recombinant DNA may be carried out 
under lower containment conditions, 
with the prior approval of the IBC and of 
NIH. 
Ill— B. Experiments with Prokaryotic 
Host- Vectors Other Than E. coli K-12 
III— B— 1. HVl dnd HV2 Systems. 
Certain certified HVl and HV2 host- 
vector systems appear in Appendix D. 
The containment levels for these 
systems are given in the subsections of 
Section III— A. Other systems in the 
future may be certified as HVl and HV2. 
At the time of certification, the 
classification of containment levels for 
experiments using them will be assigned 
by NIH. 
Ill— B — 2. Return of DNA Segments to 
Prokaryotic Non-HVl Host of Origin. 
Certain experiments involving those 
prokaryotes that exchange genetic 
information with E. coli by known 
physiological processes will be exempt 
from these Guidelines if they appear on 
the “list of exchangers” set forth in 
Appendix A (see Section I— El — 4). For a 
prokaryote which can exchange genetic 
information [35) with E. coli under 
laboratory conditions but which is not 
on the list (Host A), the following type of 
experiment may be carried out under Pi 
conditions without Host A having beer 
approved as an HVl host: DNA from 
Host A may be inserted into a vector 
and propagated in E. coli K-12 under Pi 
conditions. Subsequently, this 
recombinant DNA may be returned to 
Host A by mobilization, transformation, 
or transduction and may then be 
propagated in Host A in any desired 
vector under Pi conditions. 
For a prokaryote which does not 
exchange genetic information with E. 
coli (Host B), the following type of 
experiment may be carried out without 
Host B having been approved as an HVl 
host: DNA from Host B may be inserted 
into a vector and propagated in E. coli 
K-12 under Pi conditions. Subsequently, 
the recombinant DNA may be returned 
to Host B and propagated in Host B 
under Pi conditions. [43) 
III— B— 3. Non-HVl Systems. 
Containment levels for other classes of 
experiments involving non-HVl systems 
may be approved by the Director, NIH. 
(See Sections IV-E-l-b-(l)-(b), IV-E-1- 
b — (2)— (c), and IV-E-l-b-(3)-(b).) 
In those cases where genetic 
exchange has not been demonstrated 
between two bacterial species A and B, 
neither of which is known to be 
pathogenic for man, animals, or plants, 
recombinant DNA experiments 
involving only A and B can be 
conducted under P3 containment. [2A) 
III-C. Experiments with Eukaryotic 
Host- Vectors. 
Ill— C— 1. Vertebrate Host-Vector 
Systems. (44) (Summary given in Table 
IV). 
III-C-1-a. Polyoma Virus. 
Ill— C— 1— a— (1 ). Productive Virus-Cell 
Interactions. 
I II— C— 1— a— (1 ) — ( a ) . Defective or whole 
polyoma virus genomes, with 
appropriate helper, if necessary, can be 
used in P2 conditions to propagate DNA 
sequences: 
III— C— 1— a— (1)— (a)— (^ ). from bacteria of 
class 1 or class 2 (7) or their phages or 
plasmids, except for those that produce 
potent polypeptide toxins: [34) 
III— C— 1— a— (1)— (a)— (^). from mice: 
III— C— 1— a— {1 )— (a)— {^). from eukaryotic 
organisms that do not produce potent 
polypeptide toxins. [34) provided that 
the DNA segment is >99% pure. 
II I— C— 1 — a— ( 1 )— ( b ) . Defective polyoma 
genomes, with appropriate helper, if 
necessary, can be used in P2 conditions 
for shotgun experiments to propagate 
DNA sequences from eukaryotic 
organisms that do not produce potent 
polypeptide toxins. [34) 
III— O- 1— a— (1)— (c). Whole virus 
genomes with appropriate helper, if 
necessary, can be used in P3 conditions 
for shotgun experiments to propagate 
DNA sequences from eukaryotic 
organisms that do not produce potent 
polypeptide toxins. [34) 
III— 0—1— a— (1) — («J). Experiments 
involving the use of defective polyoma 
virus genomes to propagate DNA 
sequences from eukaryotic viruses will 
be evaluated by NIH on a case-by-case 
basis [45) and will be conducted under 
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